The conversion of a normal diploid cell to one that is malignant is a prolonged process. This conversion takes months to years in rodents and humans, respectively, and it involves widespread changes in gene expression (Bartek and Lucas, Nature 411:1001-1002, 2001; Nebert, Toxicol. 181-182:131-141, 2002). Separation of causal changes in gene expression from those that are a consequence of the malignant process is sometimes difficult; yet a causal role for tumor suppressor gene inactivation in initiating and maintaining the malignant phenotype is clear (Weinberg, Ann. NY Acad. Sci. 758:331-338, 1995). Tumor suppressor genes have a variety of functions including the control of cellular growth and the recognition and repair of DNA damage (Baylin et al., Hum. Mol. Genet. 10:687-692, 2001; Oliveira et al., Am. J. Clin. Pathol. 124:S16-28, 2005). One pathway for tumor suppressor gene inactivation is mutation (Turker, Semin. Cancer Biol. 8:407-419, 1998; Turker, Mutagenesis 18:1-6, 2003) and a second pathway is gene silencing (Fruhwald and Plass, Mol. Genet. Metab. 75:1-16, 2002; Jones and Laird, Nat. Genet. 21:163-167, 1999; Baylin et al., Adv. Cancer Res., 72:141-196, 1998; Laird, Hum. Mol. Genet. 14:R65-76, 2005). While a routine DNA sequence analysis will not reveal changes in the promoter or coding region of a silenced allele, relative to its expressed counterpart, a bisulfite sequence analysis usually reveals increased levels of promoter region DNA methylation (Chen et al., Nat. Genet. 33:197-202, 2003; Toyooka et al., Cancer Res. 62:3384-3386, 2002; Lee et al., Cancer Res. 61:6688-6692, 2001; Worm et al., Oncogene 19:5111-5115, 2000; Pogribny and James, Cancer Lett. 187:69-75, 2002). Chromatin changes are associated with promoter region DNA methylation in part because methyl binding proteins recruit repressive factors such as histone deacetylases (Bird and Wolffe, Cell 99:451-545, 1999; Ng and Bird, Curr. Opin. Genet. Dev. 9:158-163, 1999). Thus, a marker for silencing in cancer is a hypermethylated promoter combined with repressive chromatin modifications (Palii and Robertson, Crit. Rev. Eukaryot. Gene Expr. 17:295-316, 2007).
DNA methylation-associated silencing is a dominant pathway for gene inactivation in human cancers (Jones and Laird, Nat. Genet. 21:163-167, 1999). For example, silencing of critical genes such as MLH1 (Murata et al., Oncogene 21:5696-5703, 2002), BRCA1 (Esteller et al., J. Natl. Cancer Inst. 92:564-569, 2000; Matros et al., Breast Cancer Res. Treat. 91:179-186, 2005), E-CAD (E-cadherin) (Graff et al., Cancer Res. 55:5195-5199, 1995; Lombaerts et al., Br. J. Cancer 94:661-671, 2006), and ERα (estrogen receptor alpha) (Kim et al., Int. J. Mol. Med. 14:289-293, 2004; Yan et al., J. Mammary Gland Biol. Neoplasia 6:183-192, 2001) is more common than mutation in sporadic breast cancers. Based on observations such as these, one approach for treating cancer is to use pharmacological and/or dietary interventions to reactivate silenced tumor suppressor genes (Gronbaek et al., Apmis 115:1039-1059, 2007; Mund et al., Epigenetics 1:7-13, 2006). A potential flaw with this approach, however, is that chemically reactivated alleles retain epigenetic chromatin marks consistent with silenced states (Egger et al., Cancer Res. 67:346-353, 2007; McGarvey et al., Cancer Res. 66:3541-3549, 2006), which suggests that reactivated alleles frequently re-silence because they retain epigenetic “scars” of silencing. Therefore, identification of inhibitors of gene silencing, such as compounds that reduce or prevent epigenetic changes, is needed to identify potential chemopreventive agents.